SXT-R391 Integrative conjugative elements (ICEs) are self-transmissible mobile genetic elements able to confer multidrug resistance and other adaptive features to bacterial hosts, including Vibrio cholerae, the causative agent of cholera. ICEs are arranged in a mosaic genetic structure composed of a conserved backbone interspersed with variable DNA clusters located in conserved hot spots. In this study, we investigated ICE acquisition and subsequent microevolution in pandemic V. cholerae. Ninety-six ICEs were retrieved from publicly available sequence databases from V. cholerae clinical strains and were compared to a set of reference ICEs. Comparative genomics highlighted the existence of five main ICE groups with a distinct genetic makeup, exemplified by ICEVchInd5, ICEVchMoz10, SXT, ICEVchInd6, and ICEVchBan11. ICEVchInd5 (the most frequent element, represented by 70 of 96 elements analyzed) displayed no sequence rearrangements and was characterized by 46 single nucleotide polymorphisms (SNPs). SNP analysis revealed that recent inter-ICE homologous recombination between ICEVchInd5 and other ICEs circulating in gammaproteobacteria generated ICEVchMoz10, ICEVchInd6, and ICEVchBan11. Bayesian phylogenetic analyses indicated that ICEVchInd5 and SXT were independently acquired by the current pandemic V. cholerae O1 and O139 lineages, respectively, within a period of only a few years.